Anaplasma phagocytophilum, the causative agent of human Anaplasmosis, is a tick-borne pathogen with an unusual tropism for the front-line immune defense cells (neutrophils). While several aspects of the manipulation of the mammalian host cells by this gram-negative bacterium are known, relatively little is known regarding the bacterial proteins involved in exploiting the host cells to establish a successful infection. The obligate intracellular nature of this bacterium severely limits the application of conventional genetic manipulation to study the underlying virulence mechanisms. For example, it is not possible to perform targeted deletion of genes of this bacterium. In this context, we used yeast as a surrogate host to identify the virulence factors of A. phagocytophilum. Expression of bacterial products in yeast can potentially alter yeast physiology if they interfere with a eukaryotic process that is rate-limiting for growth. This system is emerging as a powerful approach to identify bacterial virulence strategies. High conservation of many (or probably most) fundamental signaling mechanisms of cell physiology between yeast and mammals, as well as the simplicity of yeast genetics are the key advantages of this system. We expressed 35 A. phagocytophilum proteins (selected by bioinformatics approach) in yeast and identified one A. phagocytophilum protein, AptA (Anaplasma phagocytophilum toxin A) whose expression severely inhibits yeast growth. AptA localizes to yeast plasma membrane, and interferes with the transport pathway involving the yeast vacuole, an organelle functionally comparable to mammalian lysosomes. This observation may have larger implications because an A. phagocytophilum containing membranous compartment does not fuse with lysosomes in mammalian cells. We hypothesize that AptA alters eukaryotic endocytotic/ vacuolar transport pathway by interfering with the function of one or more eukaryotic regulatory protein(s), and herein propose to further characterize the underlying mechanism, using the HL-60 cells, the in vitro model of A. phagocytophilum infection. The specific aims are: 1) Determine the effect of AptA on mammalian endocytic pathways. 2) Determine whether AptA is exposed (secreted or surface displayed) to the host cell. 3) Identify the eukaryotic/mammalian protein(s)/pathway(s) targeted by AptA. PUBLIC HEALTH RELEVANCE: The proposed project aims to delineate the mechanism by which a protein called AptA encoded by Anaplasma phagocytophilum, a Rickettsiales'bacterial pathogen, alters eukaryotic host physiology. Anaplasma phagocytophilum, the causative agent of human Anaplasmosis that infects neutrophils, is among the highest tick-transmitted illnesses of the USA, and hence any successful outcome of this will have significant impact on our understanding on this bacterium and potentially on the related group of Rickettsial pathogens.